7PH1

Trypsin in complex with BPTI mutant (2S)-2-amino-4-monofluorobutanoic acid

Summary for 7PH1

• Entry DOI: 10.2210/pdb7ph1/pdb
• Related: 4Y0Y 4Y0Z 4Y10 4Y11
• Descriptor: Cationic trypsin, Pancreatic trypsin inhibitor, SULFATE ION, ... (6 entities in total)
• Functional Keywords: hydrolase inhibitors, metal-binding, serine protease, hydrolase-2 inhibitor complex, hydrolase
• Biological source: Bos taurus (cattle); More
• Total number of polymer chains: 2
• Total formula weight: 31786.32

Authors

Dimos, N.,Leppkes, J.,Koksch, B.,Loll, B. (deposition date: 2021-08-16, release date: 2022-03-30, Last modification date: 2024-01-31)

Primary citation

Wehrhan, L.,Leppkes, J.,Dimos, N.,Loll, B.,Koksch, B.,Keller, B.G.
Water Network in the Binding Pocket of Fluorinated BPTI-Trypsin Complexes─Insights from Simulation and Experiment.
J.Phys.Chem.B, 126:9985-9999, 2022

PubMed Abstract: Structural waters in the S1 binding pocket of β-trypsin are critical for the stabilization of the complex of β-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly-BPTI-β-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly.

PubMed: 36409613
DOI: 10.1021/acs.jpcb.2c05496

Experimental method

X-RAY DIFFRACTION (1.18 Å)